Establishment of Human PD-1/PD-L1 Blockade Assay Based on Surface Plasmon Resonance (SPR) Biosensor

Blockade of the programmed cell death protein 1 (PD-1)/PD-ligand 1 (PD-L1) axis is a promising strategy for cancer immunotherapy. Although antibody-based PD-1/PD-L1 inhibitors have shown remarkable results in clinical cancer studies, their inherent limitations underscore the significance of developing novel PD-1/PD-L1 inhibitors. Small molecule inhibitors have several advantages over antibody-based inhibitors, including favorable tumor penetration and oral bioavailability, fewer side effects, easier administration, preferred biological half-life, and lower cost. However, small molecule inhibitors that directly target the PD-1/PD-L1 interaction are still in the early development stage, partially due to the lack of reliable biophysical assays. Herein, we present a novel PD-1/PD-L1 blockade assay using a surface plasmon resonance (SPR)-based technique. This blockade assay immobilizes human PD-1 on a sensor chip, which interacts with PD-L1 inhibitors or negative PD-L1 binders with human PD-L1 protein at a range of molecular ratios. The binding kinetics of PD-L1 to PD-1 and the blockade rates of small molecules were determined. Compared to other techniques such as PD-1/PD-L1 pair enzyme-linked immunosorbent assay (ELISA) and AlphaLISA immunoassays, our SPR-based method offers real-time and label-free detection with advantages including shorter experimental runs and smaller sample quantity requirements. Key features A SPR protocol screens compounds for their capacity to block the PD-1/PD-L1 interaction. Validation of PD-1/PD-L1 interaction, followed by assessing blockade effects with known inhibitors BMS-1166 and BMS-202, and a negative control NO-Losartan A. Analysis of percentage blockade of PD-1/PD-L1 of the samples to obtain the IC50. Broad applications in the discovery of small molecule–based PD-1/PD-L1 inhibitors for cancer immunotherapy. Graphical overview


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Published: Aug 05, 2023 Alsaab et al., 2017;Zinzani et al., 2017;Akinleye and Rasool et al., 2019). Alternatively, small-molecule inhibitors can overcome these advantages due to better tumor penetration and oral availability (Zhan et al., 2016). Therefore, the discovery of small molecule inhibitors blocking the PD-1/PD-L1 interaction is a promising cancer therapy approach. Our group reported the evaluation of the PD-1/PD-L1 blockade (using a pair-ELISA technique) and the binding of compounds to either PD-1 or PD-L1 (Li et al., 2022). However, this method is not efficient for large-scale screenings of small molecule libraries for PD-1/PD-L1 inhibitors. Therefore, we developed a surface plasmon resonance (SPR)-based PD-1/PD-L1 blockade screening approach utilizing immobilized PD-1 (on the chip), PD-L1 (in solution), and known inhibitors (i.e., BMS-1166 or BMS-202, in solution). To exclude potential false positives, we included a negative PD-L1 inhibitor (NO-Losartan A) possessing a biphenyl group-a structural feature shared with the BMS-1166 and the BMS-202 compounds that were investigated together in this study. Notably, the SPR technique is a valuable complementary method to ELISA immunoassays that can also be used for the optimization of ELISA-based assays (Vaisocherová et al., 2009). SPR is an optical biosensor technology that employs the evanescent wave phenomenon to detect changes in the refractive index of a biosensor (Pattnaik, 2005). A light source illuminates the biosensor and prism, and as the analyte flows through the channel and binds to the target protein, the refractive index of the biosensor undergoes a shift. This interaction between analyte and protein is monitored in real-time, enabling precise measurement of the amount of bound protein as well as the rates of association and dissociation. The SPR assay has unique advantages over the ELISA-type assay. Rather than merely providing an endpoint, the SPR assay monitors the kinetics associated with the PD-1/PD-L1 blockade of small molecules in real time. We acknowledge that ELISA-type assays are more widely accessible and adaptable to different laboratory settings, and we recognize that our SPR assay requires specialized instrumentation and expertise, which may not be available in all laboratories. Furthermore, given that this blockade assay is solely based on in vitro experiments, it is imperative to perform functional assays and in vivo validation to confirm the potential of compounds exhibiting blockade effects against PD-1/PD-L1. However, we believe that this SPR-based protocol, which provides sufficient details, can facilitate the screening process of small molecule inhibitors that block the PD-1/PD-L1 interaction at a large scale. In the present study, we utilized an SPR-based assay to determine the IC50 values of BMS-1166 and BMS-202, which were measured at 85.4 and 654.4 nM, respectively. BMS-1166 has been previously characterized with an IC50 value of 1.4 and 276 nM by homogeneous time-resolved fluorescence (HTRF) and cell-based assays (Jurkat cells expressing PD-1 in co-culture with CHO cells expressing PD-L1), respectively (Guzik et al., 2017). Previous investigations have reported IC50 values of BMS-202 at 18 and 96 nM utilizing different assays, including cell-based and HTRF approaches (Surmiak et al., 2021). Our results are comparable with previous findings and confirm the reliability and reproducibility of our SPR-based protocol. a. Prepare a stock solution of PD-1 at 500 μg/mL in DNase-free water (see Recipes).

Biological materials
i. Set at room temperature for 30 min to fully dissolve. ii. Dilute PD-1 solution to 40 μg/mL in acetate 5.0 and add 160 μL to tube R2 B1. b. Add 70 μL of 50 mM NaOH to tube R2 B2. c. Add 140 μL of ethanolamine to tubes R2 B3 and R2 C3. d. R2 B4 and R2 C4 remain empty. e. Add 100 μL of NHS to tube R2 B5 and R2 C5 (NHS is included in the Amine Coupling kit). f. Add 100 μL of EDC to tubes R2 B6 and R2 C6 (EDC is included in the Amine Coupling kit). 4. Place Tube A into the HBS-EP+ running buffer solution. 5. Eject the maintenance sensor chip and insert the CM5 chip. 6. Reopen the immobilization method, eject the rack, and insert reagent rack 2. 7. Run method for the estimated run time. 8. The following method will be performed: a. Inject ligand solution for five pre-concentrations. b. Establish a baseline with an injection of the HBS-EP+ running buffer solution. c. Mix inject a 50:50 ratio of EDC + NHS with a contact time of 420 s and a flow rate of 10 μL/min to activate the chip surface with the modification of carboxymethyl groups to N-hydroxysuccinimide esters. d. Continue baseline with an injection of the HBS-EP+ running buffer solution after chip modification.
The baseline activation will observe a slight response unit (RU) effect. e. Inject 40 μg/mL of PD-1 ligand to induce an electrostatic interaction that will couple the ligand to the chip surface. The ligand includes both immobilized and non-covalently bound proteins. At this stage, the PD-1 solution remains in contact with the CM5 sensor surface, resulting in a response that includes both immobilized and non-covalently bound PD-1. The N-hydroxysuccinimide esters present on the sensor chip surface react spontaneously with the primary amines on PD-1 to form stable and covalent links. f. Immobilize the ligand prior to deactivation. This indicates that the ligand has surpassed the protein surface and the majority of the non-covalently bound ligand has been removed. g. Deactivate remaining NHS-esters and remove unreacted esters through the injection of ethanolamine with NaOH utilizing a contact time of 420 s and a flow rate of 10 μL/min. The unreacted NHS-esters were deactivated using 35 μL of 1 M ethanolamine hydrochloride, which was adjusted to pH 8.5 with NaOH. Additionally, the deactivation process ensures the removal of any remaining electrostatically bound PD-1.  iii. R1 A3 as PD-L1 10 nM. iv. R1 A4 as PD-L1 20 nM. v. R1 A5 as PD-L1 40 nM. p. Set Glycine 1.5 for regeneration in the same plate layout.

B. Validation of PD-1/PD-L1 interaction
i. R1 B1-B12. q. Set sample buffer for a startup in desired well positions.
i. R1 C1-C4.  and perform a 5-fold serial dilution (20 μL) in the plate to 625, 125, 25, 5, and 1 nM. d. Add 80 μL of PD-L1 (20 nM) + HBS-EP+ running buffer + 0.01% DMSO solution to R1 A1. e. Add 3 mL of the regeneration solution (Glycine 1.5) to R2 A1. 6. Add 0.01% DMSO to HBS-EP+ to use as the running buffer. 7. Tightly cover the plate with a microplate seal. 8. Remove the previously immobilized PD-1 CM5 chip from 4 °C. 9. Eject the maintenance chip and insert PD-1 CM5 chip. 10. Place Tube A into the HBS-EP+ running buffer + 0.01% DMSO solution. 11. Open the established method, eject the rack, and insert the 96-well microplate. 12. Run method for the estimated run time. 13. After running the method, eject the chip and insert the maintenance chip. 14. Wash the chip with two drops of DI water and place at 4 °C. 15. Replace running buffer with Milli-Q water for standby.

D. PD-1/PD-L1 blockade assay with established small molecule inhibitor: BMS-202
1. Set the same method on the SPR instrument as the BMS-1166 method.

Note: In this protocol, NO-Losartan A was employed as a negative control due to the presence of a biphenyl group, which is a structural feature shared by the BMS-1166 and BMS-202 compounds tested. To ascertain the suitability of NO-Losartan A as a negative control, we conducted a preliminary investigation of its binding affinity with PD-L1. Our findings indicated that NO-Losartan A displayed negligible binding
affinity towards PD-L1, confirming its suitability as a negative control in this protocol. For screening purposes, the selection of a negative control may depend on the specific composition of the compound library employed. It is essential to ensure that the chosen negative control exhibits no binding affinity towards the PD-L1.

Data analysis
1. Immobilization of PD-1: Data were analyzed via the output from the SPR instrument indicating a low RU of the blank cell and a successful target RU of the PD-1 ligand.

Validation of PD-1/PD-L1 interaction:
Data were analyzed using the corresponding Biacore T200 analysis software (BIAevaluation version 4.1) a. Under kinetics/affinity, select surface bound. b. Select the curve as 2-1. c. Perform a 1:1 binding mode with a constant fit to obtain the association rate (Ka), dissociation rate (Kd), and dissociation constant (KD). d. Export analyzed curves into GraphPad Prism for graphical representation of data. GraphPad Prism. h. The XY analysis function was selected, and log (inhibitor) vs. response -Variable slope (four parameters) was chosen. i. The IC50 values of each compound were obtained, and a goodness-of-fit assessment was performed. A coefficient of determination (R-squared) greater than 0.99 was required for a satisfactory fit.

Validation of protocol
1. Immobilization of PD-1 on SPR chip: a. Flow cell 1 was immobilized as the blank with a final response (RU) of 103.1 ( Figure 1A). b. Flow cell 2 was immobilized with PD-1 ligand coated on the chip surface with a final response (RU) of 3688.5, indicating a successful reach of the target ( Figure 1B). The first phase (phase 1) represents a stable baseline, while the second phase (phase 2) displays a responsive effect in the response unit (RU). In phase 3, a wash with ethanolamine hydrochloride (1 M; pH 8.5) was conducted. In phase 4, recombinant PD-1 protein (40 μg/mL) was injected and coupled to the surface matrix of flow cell 2, and running buffer was injected in the flow cell 1. In phase 5, any remaining electrostatically bound ligand was removed using ethanolamine hydrochloride (1 M; pH 8.5) to deactivate unreacted NHS-esters with a contact time of 420 s and a flow rate of 10 μL/min.